Light sheet microscope with movable container

ABSTRACT

The present application discloses a light sheet microscope for imaging biological materials. The microscope uses a plurality of light beams, focused to an overlapping line to excite a fluorescent material within the biological sample. The laser-induced fluorescence image is then analyzed and displayed.

CROSS REFERENCE TO RELATED APPLICATIONS

The PCT application claims priority to U.S. Provisional Application Ser.No. 62/783,231, filed Dec. 21, 2018 and U.S. Provisional ApplicationSer. No. 62/7868917, filed Jun. 30, 2018. Each of these priorapplications is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

STATEMENT REGARDING MICROFICHE APPENDIX

Not applicable.

BACKGROUND

This invention relates to a system for imaging biological samples.

Imaging systems exist that use laser induced fluorescence to imagebiological samples.

Some of these systems can generate images of subsurface structures withimpressive clarity, precision and resolution.

Some of these systems employ F-theta scanning mechanisms, which scan asingle focused laser into the sample using a rapidly moving mirror.Because the speed of the mirror movement directly determines the imageacquisition speed, these systems are limited in the speed with whichdata can be acquired, which limits the time scale of effects they areable to capture. Also, the moving mirror has significant mechanicalcomplexity, which adds cost and makes and makes high resolutionproblematic.

Accordingly, what is needed is a microscope which images biologicalsamples in three dimensions with excellent resolution, contrast andaccuracy with few moving parts, and no high speed moving parts, that iscost effective and easy to use.

SUMMARY

An object of this invention is a microscope which has an enhancedability to precisely image internal, sub-surface structures, orstructures disposed in the bulk of a sample.

An object of this invention is a microscope which is able to removenoise in a measured image to obtain a corrected image by errorcorrection.

An object of this invention is to create three-dimensional images byscanning a light sheet laterally across a sample, and then orthogonallythrough the depth.

An object of this invention is a microscope which can have the samplechanged without affecting the excitation or detection optics.

An object of this invention is a microscope which can have the objectivelens change without affecting the excitation optics.

An object of this invention is a microscope wherein the sample can bewithdrawn from the clearing fluid without touching the sample oraffecting the excitation or detection optics.

An object of this invention is a microscope which images biologicalsamples in three dimensions with excellent resolution, contrast andaccuracy with few moving parts, and no high speed moving parts.

A light sheet microscope for imaging biological samples is described. Ina first embodiment, the microscope may include at least two collimatedlight sources each emitting a beam of light along at least two differentpropagation axes, at least two optical subassemblies which focus the atleast two beams of light into at least two straight lines, wherein atleast one of the two straight lines defines a non-orthogonal angle withrespect to its propagation axis and wherein the at least two straightlines are substantially overlapping, and wherein the straight lines andthe propagation axes define an excitation plane of the light sheetmicroscope.

In another embodiment, a light sheet microscope for imaging a biologicalsample is described, which may include at least one light source focusedby an optical assembly to a single line focus illuminating thebiological sample, defining an excitation with an intensity distributionfunction Λ₁. The excitation may cause the biological sample to emitfluorescence. The microscope may also include a means to move the singleline focus, wherein the single line focus remains in the object sidedfocal plane of the microscope, and thereby illuminating a plurality oflaterally adjacent positions in the biological sample. The microscopemay further include an imaging system that generates a two-dimensionalimage of the three-dimensional biological sample employing a mappingfunction Λ₂, and a pixelated detector. The pixelated detector mayconvert the two-dimensional images of the microscope of the fluorescenceemitted by the sample to at least two raw images, wherein the pixelateddetector has a point spread function Λ₃, and a computer that can storeand manipulate the signals produced by the pixelated detector and isprogrammed to produce a restored pixelated image F from the raw images Iby removing degradations associated with the functions Λ₁, Λ₂ and Λ₃.

In other embodiments, a light sheet microscope for imaging a firstbiological sample disposed on a sample holder is disclosed. In thisembodiment, the microscope may include an imaging lens structureincluding an operative objective lens having a focal plane and at leastone inoperative lens, and wherein the imaging lens structure is movablein a z-direction orthogonal to the focal plane by a movable first stage.The microscope may further include a container holding a quantity offluid, wherein the sample holder is immersible in the fluid, and adetector which forms an image of the focal plane, wherein the imageincludes at least a portion of the first biological sample. The movablefirst stage supporting the lens structure may have sufficient range ofmotion to submerge the operative objective lens into the fluid held inthe container, thereby forming an image of the first biological sampleon the detector, and wherein the sample holder. The container may have ashape which admits a movement of the imaging lens structure, when theimaging lens structure is submerged.

In yet other embodiments, a light sheet microscope for imaging abiological sample, located on a sample holder is disclosed. Themicroscope may include a detector which forms an image of the biologicalsample through imaging optics, wherein the biological sample disposed ina focal plane of the imaging optics. The microscope may further includea container holding a quantity of fluid and disposed on a movable firststage, movable in the z-direction, wherein the z-direction is orthogonalto the focal plane, a sample holder holding the biological sample.Within this embodiment, the biological sample may be immersed in thefluid and the biological sample may be in the focal plane, wherein thefirst stage has a range of motion such that the sample can be bothimmersed in the fluid and in the focal plane and then withdrawn from thefluid by the motion of the first stage, wherein the first stage movesthe container independently of the sample holder, the imaging optics andthe detector.

These and other features and advantages are described in, or areapparent from, the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary details are described with reference to the followingfigures, wherein:

FIG. 1 is an illustration of a line focus in a biological imagingdevice, wherein the line focus is tilted with respect to an axis ofpropagation of radiation;

FIG. 2 is an illustration of a two overlapping line foci in a biologicalimaging device with two axes of propagation of radiation;

FIG. 3 is an illustration of a three overlapping line foci in abiological imaging device with three axes of propagation of radiation;

FIG. 4 is an illustration of a light sheet microscope with three axes ofpropagation of radiation, showing the lateral capability;

FIG. 5 is an illustration of a three overlapping line foci in abiological imaging device with three axes of propagation of radiation,having a rotatable slot structure that may define a numerical apertureof the imaging system;

FIG. 6a is a perspective view of the rotatable slot structure that maydefine a numerical aperture of the imaging system; FIG. 6b is a crosssectional view of the rotatable slot structure;

FIG. 7 is an illustration of the intensity of the line focus as afunction of the lateral distance showing two successive scans, anddemonstrating Gaussian propagation of the beam;

FIG. 8a is an illustration of the intensity of the line focus as afunction of lateral distance, and an illustration of the beam waist andRayleigh distance for a first numerical aperture NA₁; FIG. 8b is anillustration of the intensity of the line focus as a function of lateraldistance, and an illustration of the beam waist and Rayleigh distancefor a second numerical aperture NA₂, wherein NA₁<NA₂;

FIG. 9 is an illustration showing the lateral horizontal movement of thelight sheet within the biological sample;

FIG. 10a , FIG. 10b are two alternative arrangements of the opticalturning elements that can generate the lateral horizontal movement ofthe light sheet within the biological sample for the imaging device;

FIG. 11 is an illustration showing the left side and right side lateralhorizontal movement of the light sheet within the biological sample,which may image the sample from the left and from the right,respectively;

FIG. 12 is an illustration describing the correction for refraction ofthe light beams at the boundary of the glass liquid vessel;

FIG. 13 is an illustration of a light sheet microscope with a pixelateddetector imaging a sample within its field of view of the detector;

FIG. 14 is an illustration showing the point spread functions of theexcitation Λ₁ and the detection Λ₂, Λ₃ produced by the light sheetmicroscope from the biological sample;

FIG. 15 portrays the different steps (FIG. 15a , FIG. 15b , FIG. 15c ,FIG. 15d ) of image formation;

FIG. 16 is an illustration describing how the left side of the image ismeshed with the right side image using the device shown in FIG. 11;

FIG. 17 is an illustration showing the lateral horizontal scanning ofthe light sheet through the biological sample in the x-direction;

FIG. 18 is an illustration showing horizontal scans (FIG. 18a ,x-dimensions) and scanning in depth (FIG. 18b , z-dimension) to producea three dimensional image by the light sheet microscope from thebiological sample

FIG. 19 is an illustration showing the filter selection to choose aworking wavelength for the light sheet microscope excitation anddetection;

FIG. 20 is an illustration showing the plurality of optical sources(FIG. 20a and FIG. 20b ) for the light sheet microscope excitation anddetection;

FIG. 21 is an illustration showing the components of the detector forthe light sheet microscope;

FIG. 22a is an illustration showing the ability to rotate a turretholding a plurality of objective lenses; FIG. 22b is a perspectiveillustration of the sample stage and turreted lenses, accommodating theturreted lenses; FIG. 22c is a perspective illustration of the turretedlenses;

FIG. 23 is an illustration showing how the light sheet may be movedlaterally independently of the turreted objective microscope;

FIG. 24 is an illustration showing a view of how the cuvette may beimmersed by vertical movement, (FIG. 24a ) independently of the sampleand the optical system (FIG. 24b );

FIG. 25a plan view showing the movable cuvette; FIG. 25b is a crosssectional view, and FIG. 25c is an end-on view how the cuvette andsample holder may be moved vertically independently of the sample andthe optical system;

FIG. 26a is a side view illustration showing how the sample may beaccessed without disturbing the rest of the optical system; FIG. 26bshows the sample completely withdrawn from the clearing fluid;

FIG. 27 is an illustration showing how multiple samples may be handledusing the novel sample holder.

It should be understood that the drawings are not necessarily to scale,and that like numbers may refer to like features.

DETAILED DESCRIPTION

The first portion of this description is directed to the optical detailsof the novel optical imaging device for biological samples using lightsheets, and error correction. The device also has some design featuresthat make it remarkably simple and easy to use. The novel imaging devicemay reduce the uncertainly deriving from the shadows cast by opaquestructures, and also has no high speed moving parts and so may beconsiderably simpler than other scanning methodologies. The secondportion discusses the error correction methodology used to improve theimage data in terms of accuracy, contrast and resolution. The thirdportion describes some of the novel mechanical features of the lightsheet microscope that make it particularly advantageous and easy to use.

In the following description of the preferred embodiment, reference ismade to the accompanying drawings which form a part hereof, and in whichis shown by way of illustration a specific embodiment in which theinvention may be practiced. It is to be understood that otherembodiments may be utilized and structural changes may be made withoutdeparting from the scope of the present invention. The followingreference numbers are used to refer to the following features. It shouldbe understood that this list is provided as a convenience, and may notbe exhaustive of the reference numbers used in the text that follows.

-   -   10 Lens turret    -   60 Turning mirrors including 61, 62 and 63    -   61, 62, 63 Turning mirrors embodiment 100    -   68, 64, 66, 74 Turning mirrors in alternative embodiment    -   65, 67, 69 Turning mirrors embodiment 101    -   74 First turning mirrors embodiment 100, 101    -   51 rotating aperture including slot 73 and aperture 52    -   68, 69 turning mirrors    -   70 telescoping lens    -   73 slot    -   100 Movable optical assembly    -   101 Alternative embodiment of optical system    -   100′ Adjacent side movable optical assembly    -   120 Line focusing optical subassembly #1    -   140 Line focusing optical subassembly #2    -   160 Line focusing optical subassembly #3    -   200 Biological sample    -   210 Line focus/focal plane    -   300 Cuvette    -   310 Field of view of detector    -   350 Top surface opening for objective lens    -   351, 352 Cutouts from cuvette 300    -   360, 361 Transparent windows for admitting radiation to the        cuvette and sample    -   400 Controller    -   500 light source    -   521-526 Laser sources    -   530 Collimating lens    -   550 Filter wheel excitation    -   560 collimator    -   600 Detector    -   660 Filter wheel detector    -   620 imaging lens    -   660 detection filter    -   700 objective lens    -   750 particle    -   670 objective lens    -   800 sample holder    -   810 sample stage    -   820 sample stage support point    -   930 movable cuvette stage    -   900 stage for movable optical assembly    -   951, 952 Cutouts from cuvette 300    -   1000 clearing fluid

A coordinate system applies to the figures in general. The x-axis isgenerally the scanning dimension, that is, it is the axis along whichthe line focus will be scanned. The y-axis is generally the direction ofthe line focus, that is, it is the direction that the focus lies along,with relatively uniform intensity within and along the focal line. Theradiation beams entering the sample are traveling along an x-axis in thex, y plane. The z-axis is generally the viewing direction. That is, theoptical axis of the camera and/or detector will lie above and orthogonalto the x- y- plane, along the z-axis.

In one aspect, the light sheet microscope may make use of Scheimpflugoptics, which is the phenomenon whereby tilting an optical element withrespect to its optical axis, the focusing properties of that element maybe tilted as well. By careful placement and relative orientations, threeexcitation light sources may be arranged to create a single line focusthat can then be scanned across the sample.

Accordingly, at least one optical element may be tilted with respect topropagation direction by an angle of < >90 degrees (non-orthogonal).When the angle α₁ is defined relative to the line orthogonal to thepropagation axis (see FIG. 1), the angle α₁ may be less than 30 degreespreferentially, more preferably less than 20 degrees and more preferablyabout 16 degrees. As discussed below, this angle may be corrected forrefraction effects occurring at the liquid cuvette holder boundary. Thisresults in a tilt of the line focus with respect to the axis ofpropagation through the optical system by and angle α₁. α₂ may besimilar in magnitude but in the opposite sense as α₁.

According to one aspect of the invention, using the light sheetmicroscope as described herein, shadows cast by a particle are reducedor at least rendered unambiguous. Even more precision may be obtainedusing the error correction process described herein. In this process, anumber of distinct shadows, which are switchable in this case byswitching illumination sources, can be easily calculated out compared toa scanned beam, which creates one smooth shadow.

According to another aspect of the invention, a column of pixels can betreated in generally the same way in the image processing algorithm,because illumination in this dimension is uniform because of theproperties of the line focus.

According to another aspect of the invention, very high precision threedimensional images may be produced using very few moving parts, and nohigh speed moving parts. This greatly improves repeatability, cost andreliability.

Light Sheet Microscope

FIG. 1 is an illustration of a line focus used in a biological imagingdevice, wherein the imaging device uses a single collimated light source500. Radiation from the source 500 is focused into a line focus that istilted with respect to an axis of propagation of radiation. Opticalradiation from a source 500 is shaped by an optical subassembly 120, andfocused into a line 210 that falls within a biological sample 200, butis tilted with respect to the axis of propagation through the opticalsubassembly 120.

The optical subassembly 120 may contain a plurality of optical elements,including for example two confocal or spherical lenses and a cylindricallens. At least one of the lens elements may be tilted with respect tothe axis of propagation of the radiation. The tilt angle is denoted byα₁ wherein α₁ may be in the range 0<α₁<40°, and more typically in therange 5<α₁<25°, and more preferably about 16°. This tilt may result inthe line focus being tilted by a similar angle α₂ with respect to thepropagation axis of the radiation. The propagation axis or the axis ofpropagation is the direction traveled by photons at or near the centerof the beam of collimated light. The optical axis is often the same orparallel to the propagation axis but is defined as the neutral axis ofoptical elements: the line passing through the center of curvature oflens and parallel to the axis of symmetry is the optical axis. Theoptical axis of the pixelated detector, for example, is the line from afirst lens or transparent window on the front of the camera or detector,to the pixelated detector array at the rear of the camera or detector.These angles are defined relative to the axis orthogonal to the axis ofpropagation as shown in FIG. 1.

In the embodiment shown in FIG. 1, the second confocal lens (the thirdoptical element in optical subassembly 120) may be tilted by the anglewith respect to axis of propagation. The angle it forms, α₁, is measuredrelative to the orthogonal direction with respect to this propagationaxis. As a result of this tilt, the line focus 210 is also tilted by anangle α₂ with respect to this orthogonal axis. Based on the geometryshown in FIG. 1, α₁ may be similar in magnitude but opposite in signwith respect to this orthogonal axis. This is the basis of Scheimpflugoptics. The Scheimpflug principle is a geometric rule that describes theorientation of the plane of focus of an optical system (such as acamera) when the lens plane is not parallel to the image plane. Thisprinciple is used here to form overlapping lines in an biologicalsample, as described in detail below.

The elements of the optical subassembly 120 may be arranged in a numberof different ways, such as cylindrical/spherical/spherical orspherical/spherical/cylindrical. However, the configuration ofspherical/cylindrical/spherical (shown in FIG. 1) may result in the mostcompact arrangement.

Within the optical subassembly 120, two lenses may be glued together.Scheimpflug conditions can also be realized with more and gluedelements. However the configuration shown in FIG. 1 may be mostadvantageous for both Scheimpflug and curvature correction. Separatelenses also give an additional degree of freedom, because the distanceof separation may affect the shape of the line focus and its curvature.

FIG. 2 is an illustration of another embodiment of two opticalsubassemblies 120 and 140. The components of the optical subassembliesmay be similar or identical, each containing for example, two confocallenses and a cylindrical lens. The arrangement of these components maybe similar to that illustrated in the embodiment shown in FIG. 1.However this is exemplary only and the optical assembly may havedifferent components or may they may be in a different arrangement ororder.

The second optical subassembly 140 may also create a line focus, howeverthis optical assembly may not have a tilted element. Accordingly, theline focus may not be tilted with respect to the image plane, but mayinstead lie nearly exactly along the orthogonal direction, that is, itmay lie in the image plane.

It should be noted that optical subassembly 120 may be tilted withrespect to optical subassembly 140. In other words, the optical axis andof optical subassembly 120 may form an angle with respect to the opticalsubassembly 140, and the propagation axis of the light traveling throughit. Additionally, optical subassembly 120 may form a line focus at anangle with respect to its optical axis, and the propagation axis of thelight traveling through it. By proper arrangement of the components ofoptical subassembly 120 and optical subassembly 140, the line focuscaused by optical subassembly 120 may fall substantially exactly overthe line focus resulting from optical subassembly 140. These overlappingline foci may be applied to a biological sample within a biologicalimaging device with two axes of propagation of radiation.

By “substantially overlapping” or “substantially exactly overlapping”,it should be understood that the line foci deviate in overlap from oneanother by less than a defined amount. This amount may be in lateralmisregistration or angular deviation. That is, the line foci althoughsubstantially exactly overlapping, may nonetheless deviate a finiteamount laterally and angularly. The amount of lateral deviation allowedwhile still “substantially overlapping” or “substantially exactlyoverlapping”, may be defined in terms of Rayleigh length. The amount ofangular deviation may be defined in terms of degrees of angulardeviation. For the purposes of this description, “substantiallyoverlapping,” the overlapping line foci will deviate from one another byless than 4 Rayleigh lengths laterally and less than 5 degreesangularly. More preferably, the “substantially overlapping” line fociwill deviate from one another by less than 2 Rayleigh lengths and 3degrees. Yet more preferably, “substantially overlapping” or“substantially exactly overlapping”, overlapping foci may deviate fromone another by less than 1 Rayleigh length and less than 2 degrees.

“Partially overlapping” may be understood to mean the placement of oneline focus within 5 Rayleigh lengths of the adjacent line focus.

The biological sample 200 may have biological structures which aretagged with a fluorescent moiety. Accordingly, biological sample 200 mayfluoresce when radiation from source 500 having the proper wavelengthexcites these fluorescent moieties. This fluorescence may be detected byan appropriate detector and used to gain information about thebiological sample. The device is described in detail below.

Importantly, therefore, the device shown in FIG. 2 may use light 500coming from multiple directions. The different directions may eachderive from an independent light source, or they may use the same lightsource but split off the single source by partially transmitting andpartially reflecting surfaces. These configurations are also describedin further detail below.

Having radiation coming from different directions but with overlappingline foci may have several advantages. One advantage may be that shadowscast by an opaque structure intercepting light from one source may bedistinguished from other sources of contrast. Accordingly, the contrastseen in an image may be attributed to an obscuring structure, and thedetailed morphology of that structure may be ascertained by comparingthe images collected using the radiation coming from multipledirections. Accordingly, successive scanning with the off-center optics120 and 160 may also reveal the depth and extent of shadowing from asingle opaque structure in the biological sample, Accordingly, thistechnique can be used to ascertain what features in a scan are due toshadowing effects, an what features are related to real, new or discretestructures within the sample.

FIG. 3 is an illustration of a three overlapping line foci in abiological imaging device with three axes of propagation of radiation.FIG. 3 shows three optical subassemblies, 120, 140 and 160. Thecomponents of the three optical subassemblies 120, 140 and 160 may besimilar or identical, each containing for example, two confocal lensesand a cylindrical lens. The arrangement of these components may be thesame as illustrated in the embodiment shown in FIG. 1. However this isexemplary only and each optical subassembly may have differentcomponents or may they may be in a different arrangement or order.

Once again, the second optical subassembly 140 may create a line focus,however this optical assembly may not have a tilted element.Accordingly, the line focus may not be tilted with respect to the imageplane, but may instead lie nearly exactly along the orthogonaldirection, that is, it may lie in the image plane.

It should be noted that third optical subassembly 160 may be tilted withrespect to optical assemblies 120 and 140. In other words, the opticalaxis of optical subassembly 160 may form an angle with respect to theoptical axis of optical assemblies 120 and 140, and the propagation axisof the light traveling through it. Additionally, optical subassembly 160may form a line focus at an angle with respect to its optical axis. Byproper arrangement of the components of optical subassembly 160 andoptical assemblies 120 and 140, the line focus caused by opticalsubassembly 160 may fall substantially exactly over the line fociresulting from optical assemblies 120 and 140. Accordingly, in FIG. 3,there may be three substantially overlapping line foci, all designatedby reference number 210, because they are substantially overlapping.

Accordingly, the third optical subassembly 160 creates another linefocus that falls exactly on the first two line foci from opticalsubassembly 120 and 140. This third optical subassembly 160 may be themirror image of optical subassembly 120, with its tilted optical elementtilted in the mirror image sense, as shown in FIG. 3. Accordingly, theremay be a symmetry axis as depicted in FIG. 3, with the optical top half120 mirrored by the optical bottom half 160.

The overlapping line foci may be applied to a biological sample 200within a biological imaging device with three axes of propagation ofradiation. As before, the biological sample 200 may have biologicalstructures which are tagged with a fluorescent moiety. Accordingly,biological sample 200 may fluoresce when radiation from source 500having the proper wavelength excites these fluorescent moieties. Thisfluorescence may be detected by an appropriate detector and used to gaininformation about the biological sample. By applying the radiation fromthree different directions, the shadowing effects of opaque structurescan be effectively measured, such that detailed information about themorphology of the structure may be ascertained. If this line focus isthen scanned laterally through the sample, detailed information aboutstructures contained in the sample, but separated laterally, may beobtained. The means and methods for moving the line focus through thesample are described in considerable detail below.

If the light sources coming from the three directions 120, 140 and 160are activated sequentially rather than in unison, the shadows may beunambiguously detected. This is because the shadow as cast from anopaque object illuminated by a light source impinging from onedirection, will obscure a different area directly behind the opaqueobject than a shadow cast by a light source coning from anotherdirection. Accordingly, in some embodiments, the light sources 500 maybe energized sequentially, at least during a portion of the datacollection.

Finally, by energizing the three beams sequentially, a shadow cast by astructure embedded in the sample can easily be distinguished from ashadow cast by, for example, a lens or mirror defect. Furthermore, thisstructure can also be measured in extent by sequential irradiation fromthe three beams. Because of the different trajectories of the threebeams, shadows cast by a particle, by using three beams may be reducedor separated. Even further, by deconvolving a number of distinctshadows, which are switchable in this case, the effect of the shadowscan be more easily calculated compared to a scanned beam, which createsone smooth shadow.

FIG. 4 is an illustration of a three overlapping line foci in abiological imaging device with three axes of propagation of radiation,wherein the biological sample is contained in a fluid vessel and imagedby a pixelated detector. The biological sample 200 may be submerged in acuvette or other vessel 300 containing a fluid. “Cuvette” is a term usedhere to refer to a vessel containing sample embedded in various opticalliquids. The cuvette may have at least one window which is opticallytransparent with low distortion to allow for diffraction limited lightsheet generation. Thus the terms “cuvette”, “vessel” and “container” areused interchangeably to refer to a holder or receptacle that contains aquantity of fluid. The fluid may be a clearing fluid in which abiological sample is immersed. The cuvette, vessel or container may alsobe open on one side for immersion of the biological sample 200 and fordipping of the objective lenses into the fluid, as will be describedfurther below. A “clearing fluid” is a fluid that renders a biologicalsample relatively transparent to a probing radiation.

“Lateral” or “laterally adjacent” to a point should be understood to bedefined with respect to a plane through a sample, wherein a second pointlaterally adjacent generally is in the same plane but offset from afirst point also in the plane. An “anamorphic lens” may be a lens whosefocal distance in one dimension is different that its focal distance inthe other dimension. A cylindrical (line focusing) lens is one exampleof an anamorphic lens, having a first and a second the focal distance,wherein the first focal distance is finite, and the second focaldistance is essentially infinite. Such a lens will produce a line focusat the first focal distance.

A “clearing fluid” is a biological fluid containing compounds designedto minimize non-uniform light absorption or scattering by biologicalstructures in the sample. The use of clearing fluids is important inimaging into the depths of thicker biological sample, such that theprobing radiation is able to penetrate into the depth. Hydrogenperoxide, for example, can be used as a clearing fluid to de-colorhemoglobin and myoglobin, two of the primary molecules responsible forlight absorption in biological tissue.

The fluid in the cuvette 300 may be a clearing fluid 1000, which mayrender the biological tissue transparent to radiation. The cuvette 300may also be transparent, or may at least have at least one transparentwindow or opening on its side, allowing the optical radiation from thesource 500 to pass into the cuvette 300, into the fluid and into thebiological sample. 200. The lateral distance “A” to “B” may indicate thethickness of the transparent walls of the cuvette. Refraction effectsmay occur at this boundary, and the treatment of this refraction isdiscussed below with respect to FIG. 12.

As mentioned previously, the plurality of optical assemblies 120 and 140(and 160 if present) may be configured such that each focuses incomingradiation into a line. The line focus due to optical subassembly 120 and160 may substantially overlap the line focus due to optical subassembly140. Accordingly, all optical assemblies 120, 140 and 160 may focusradiation into the same line focus 210 that falls within the biologicalsample 200.

Above the cuvette 300 (and not shown in FIG. 4), a detector may imagethe sample 200 in order to detect fluorescence emitted by thefluorescent tags, for example, when irradiated by the line focus 210.The biological sample 200 may be placed in the horizontal plane (planeof the paper) and the detector may be placed above this plane. The linefocus 210 may also fall substantially in this plane. The detector mayhave field of view 310 that includes the line focus 210 in the sample,and may also include laterally adjacent areas. That is, the field ofview of the detector may include the line 210 in its first position aswell as subsequent adjacent positions, as the line focus is scannedlaterally. The lateral movement of the line focus is described furtherbelow with respect to FIG. 9.

FIG. 5 is an illustration of a three overlapping line foci in abiological imaging device with three axes of propagation of radiation,wherein the illustration includes additional optical elements thatdirect the radiation onto the sample 200. In one embodiment shown inFIG. 5, the radiation may come from a single source 500, and may bedirected off three partially reflecting and partially transmittingmirrors 61, 62 and 63. Each of these mirrors may direct a portion of theradiation into parallel lines. These parallel lines may pass through anaperture 51 described further below and then proceed in a parallelfashion to a movable optical assembly 100. FIG. 5 does not show thecomponents of subassemblies 120, 140 and 160. The important feature isthat light entering movable optical assembly 100 containingsubassemblies 120, 140 and 160 enters the movable assembly 100 in aparallel fashion, such that movable assembly 100 may be moved laterallywithout affecting the optical paths within movable assembly 100. Thisfeature is important in moving the line focus laterally through thesample, and allows the high resolution, highly repeatable scanningcapabilities of the light sheet microscope.

Accordingly, movable optical assembly 100 may contain opticalsubassemblies 120, 140 and 160 as was shown in FIG. 3. In addition,movable optical assembly 100 may also contain two pairs of turningmirrors 53, 54, 55 and 56. The first pair of turning mirrors 53 and 54may redirect the parallel incoming light and direct it into opticalsubassembly 120 along its optical axis. The second set of turningmirrors 55 and 56 may redirect the lower leg of incoming parallel lightinto optical subassembly 160 along its optical axis. Accordingly, onceagain, the lower legs are the mirror-image of the upper legs, reflectedacross the symmetry axis shown.

Because the light incoming to movable optical assembly 100 is parallel,the movable optical assembly 100 can be moved laterally without changingthe angles of beam propagation within the movable optical assembly 100,or the focusing properties thereof. The lateral motion will, as aresult, move the line focus laterally, such that the line may be scannedleft and right to move the line foci laterally within the sample. Thus,the scanning direction may be lateral, in the plane of the paper. Thatis, movable optical assembly 100 may be scanned horizontally (in theplane of the paper) in order to shift the line focus 210 laterallythrough the sample. This function is described more thoroughly withrespect to FIG. 9.

An optical element 51 is also shown in FIG. 5. This component may be arotatable component 51 which may be equipped with a number of apertureshaving a certain shape that pass the incoming radiation and define itsoptical properties. The slotted aperture 51 is shown in more detail inFIG. 6. For example, the slotted aperture 51 may define the opticalnumerical aperture of the system, as described in detail below withrespect to FIGS. 6, 7 and 8. As indicated, the slotted aperture 51 maybe rotated quickly to adapt the numerical aperture to different valueswithin the device.

The rotatable aperture 51 may be used to select the Gaussian beamproperties. In particular, as the rotatable aperture is rotated tointercept a larger portion of the beam and allow a smaller portion topass, has the effect of defining a smaller numerical aperture to thesystem.

The rotatable aperture 51 may also be able to block or disable one, twoor all three of the beams of light being reflected from any of theturning mirrors 61, 62 or 63. This selection may be performed byrotating the rotatable member 51 to a position where one, two or threeof these parallel light beams is blocked. The rotatable member 51 maygenerally be oriented in a direction perpendicular to optical axes ofthe beams reflected off turning mirrors 61, 62 and 63 and thus can beused to select (or turn off) any of 61, 62 or 63.

Also shown in FIG. 5 is a coordinate system that applies to this figureand the figures in general. The axes in the coordinate system, x, y andz, are orthogonal with respect to one another. The x-axis is generallythe scanning dimension, that is, it is the axis along which the linefocus will be scanned. The y-axis is generally the direction of the linefocus, that is, it is the direction that the focus lies along, and alongwhich the radiation intensity is relatively uniform. However, it shouldalso be understood that the scanning of the line focus may also beperformed in the y-direction, that is, in the same direction as thelength or extent of the line, rather than perpendicular to thisdimension. The z-axis is generally the viewing direction. That is, thecamera and/or detector will lie above the x- y-plane, along the z-axis.It should be understood that these orientations are arbitrary, as aredesignations such as “left,” right,” “up,” “down,” and “top,’ and“bottom” and refer only to opposing or obverse sides. The device may beheld in any orientation without loss of generality.

FIG. 6 shows the rotating slotted aperture 51 in greater detail. Asshown in FIG. 6, the rotating member 51 may be formed with a pluralityof rectangular through holes 52 formed therein. The rotating member 51may further be equipped with a plurality of intersecting slots 73. Byrotating these through holes 52 and slots 73, the clearance available toa ray of light is expanded or diminished. In other words, a shadow iseffectively cast by the incoming aperture which serves to stop down thelight transmitted through the structure. This effect is shown in thecross section of FIG. 6b . Because of the blocking effects of theapertures in the rotating slotted aperture 51, The radiation may berestricted in the z-direction, thus defining the numerical aperture ofthe system when the beam is focused into a line. These effects aredescribed further below.

The rotating member 51 may be adjusted quickly by a single motorizedaxle, which may rotate the rotatable member 51. Because the plurality ofapertures may be provided for each of the plurality of beams, theoptical properties of the overall system may be defined quickly andinexpensively using a single actuator or a rotating stepper motor, forexample. The turning mirrors 61, 62 and 63 may direct the radiation intothe appropriate aperture 52 or slot 73.

The cross section shown in FIG. 6b shows the effect of rotating therotatable component 51. As the member 51 is rotated, the leading edge ofthe aperture 52 obscures the beam path, blocking the upper portion ofthe beam from being transmitted through the aperture 52. This thentruncates the downstream portion of the beam to a narrower portion thanwas incident on the rotatable member 51. The effect may be to decreasethe numerical aperture of the optical system.

As illustrated in FIG. 6, the rotating member 51 may be a cylinder withcutouts for slots, and the cylinder may be rotated to define numericalaperture of the beam. By rotating the slotted post 51 as a unitary body,the beams sizes for all the three of the laser beams coming off turningmirror 61, 62 and 63 may be defined with one action. In other words, asingle motorized stage or stepper motor (not shown) may be required todefine the numerical aperture for all three beams. Because of itscylindrical geometry, the rotating member 51 may have little rotationalinertia so beam switching can be very rapid. The slots that obscureparts of beam may also eliminate non-uniform fringes. A telescopingcylindrical lens 70 may determine the image size and illumination area,as will be described below with respect to FIG. 9. As with many of theoptical holders and stages, the rotating member may be formed ofanodized aluminum, by machining for example.

It should be noted that the beam path lengths from turning mirrors 61,62 and 63 are all different, as shown in FIG. 5. Accordingly, the lightsource 500 may need a limited coherence length to avoid interferometriceffects such as constructive or destructive interference. Within therotatable aperture 51, wedged plates (not shown) may be moved to reducecoherence time below camera exposure.

In another embodiments, a set of simple adjustable slits may be usedwhich may be independently adjustable. Use of the slits or some otherselectable aperture may also reduce the amount of radiation applied tothe sample so reduces heating and bleaching but primarily determines thenumerical aperture and thus the quality of the imaging (resolution, stepsize, etc.). However, by putting the features all on a single post makesthe performance and optical attributes selectable by a single actuatoror motor.

Many beams such as laser beams emitting in the TEM 00 mode have beamprofiles that have a Gaussian intensity distribution. This may be thecase with the optical system described here. FIG. 7 illustrates the beamintensity versus lateral position within the beam. The shape of theintensity curve shown in FIG. 7 is intended to show a generally Gaussiancurve, wherein the width of the profile is characterized by theparameter b. The parameter b on the curve indicates the point at whichthe intensity drops to 1/sqrt(2) of the peak intensity. As is usual, thebeam reaches peak intensity near the center of the distribution, anddrops off with some characteristic shape to a value 1/sqrt(2) at adistance b from the center. b is referred to as the confocal parameter.

The geometric dependence of the fields of a Gaussian beam are governedby the wavelength λ of the radiation (in the dielectric medium, not freespace) and the following beam parameters, all of which are connected asdetailed in the following sections. The Gaussian beam width w(x) is afunction of the distance along the x-direction in which the beampropagates. W₀ is the beam waist and b is the depth of focus. Theproperties of Gaussian beams are well known, and these properties aresummarized here in order to introduce parameters that will be referredto in the discussion of the design and operation of the system, whichfollows.

The shape of a Gaussian beam of a given wavelength λ is governed solelyby one parameter, the beam waist w₀. This is a measure of the beam sizeat the point of its focus (x=0 in the above equations) where the beamwidth w(x) (as defined above) is the smallest (and likewise where theintensity on-axis (r=0) is the largest). From this parameter the otherparameters describing the beam geometry are determined. This includesthe Rayleigh range x_(R) and asymptotic beam divergence θ. Thesequantities are illustrated in FIGS. 8a and 8 b.

The Rayleigh distance or Rayleigh range x_(R) is determined given aGaussian beam's waist size. Here λ is the wavelength of the light in themedium of propagation. At a distance from the waist equal to theRayleigh range x_(R), the width w of the beam is larger than it is atthe focus where w=w₀, the beam waist. That also implies that the on-axis(r=0) intensity there is one half of the peak intensity (at x=0). Thispoint along the beam also happens to be where the wavefront curvature(1/R) is greatest. The numerical aperture of a Gaussian beam is definedto be NA=n·sin θ, where n is the index of refraction of the mediumthrough which the beam propagates and θ is the divergence angle. Thismeans that the Rayleigh range is related to the numerical aperture byx_(R)=w₀/NA. The distance between the two points x=±x_(R) is called theconfocal parameter b, or depth of focus of the beam. Gaussian beams aredescribed in, for example, https://en.wikipedia.org/wiki/Gaussian_beam.

Accordingly, the numerical aperture NA˜2w₀/b. The smaller the NA, thelarger the confocal parameter for a given beam waist. If more resolutionis needed, the confocal parameter b must be smaller so the NA must belarger and the depth of field shorter. These properties may be definedby the position of the rotatable member 51 in selecting a numericalaperture. If instead, better contrast is needed and extended focallength, to get better contrast over field of view, a lower magnificationand larger confocal parameter b may be selected (smaller NA). The NAaperture may also be tailored to increase the useful area of thedetector. Accordingly, the rotatable member 51 may be used to extendfocal length, to get better contrast over field of view by lowermagnification (lower NA), and to increase the useful area of thedetector. The rotatable member 51 may be used to rapidly adjust thewidth and numerical aperture of the system.

Accordingly, the effect of a tighter focus is a shorter beam waist andlarger divergence angle θ, in other words, a shorter depth of focus andhigher resolution. Tighter focusing is associated with a largernumerical aperture NA. Accordingly, a larger numerical aperture NAimplies a smaller confocal parameter b. Thus a more tightly focused beamcan give better resolution but over a shorter distance.

FIG. 7 is an illustration of the intensity of the line focus as afunction of the lateral distance from the center of the focus. A typicallight intensity profile for successive scans, may generally have aGaussian shape and follow Gaussian optics as discussed above. However,other functions may also be used, such as for example, Bessel andLagrangian shapes.

The width of the line focus b and separation Δx between scans may berelated and chosen based on attributes of the sample (size, thickness,density, etc.), and based on performance considerations. In general, thehigher the resolution, the smaller the step size and the longer the timerequired to complete a scan. In addition, the sample will be subjectedto higher excitation intensities and thus higher temperatures, bleachingand photodamage. In other words, these variables may be related, suchthat design tradeoffs may need to be made. A given choice of numericalaperture may determine the step size and resolution of the scannedimage. These parameters and design tradeoffs are discussed further belowwith respect to FIGS. 12 and 13. The choice of NA which determines thefocusing attributes may also determine step size, acquisition speed, andother important system level operational attributes.

FIG. 8a is an illustration of the intensity of the line focus as afunction of lateral distance, and an illustration of the beam waist andRayleigh distance for a first numerical aperture NA₁. FIG. 8b is anillustration of the intensity of the line focus as a function of lateraldistance, and an illustration of the beam waist and Rayleigh distancefor a second numerical aperture NA₂, wherein NA₂>NA₁. As mentionedpreviously, the Rayleigh length is determined by the waist radius wo andthe wavelength λ.

There can thus be a trade-off between a more strongly focused beam withhigher optical intensity in the focus, and a less strongly focused beamwith longer Rayleigh length, i.e. larger depth of focus. The reduced NAmay be perpendicular to light sheet. Choosing a wider slot increases thenumerical aperture of the beam, which decreases the confocal parameter band increases the resolution. Conversely, choosing a narrower slot maydecrease the confocal parameter b, and reduce the resolution. Asmentioned previously therefore, the choice of numerical aperture maydrive the spacing between scans Δx and thus the speed of imageacquisition and maximum sample irradiation intensity. In view of this,the choice of numerical aperture chosen with the rotating member 51 maybe a central design choice.

FIGS. 9 and 10 are embodiments exemplifying an important aspect of thelight sheet microscope described here. This aspect is the ability of themovable optical assembly 100 and 101 to move laterally with respect tothe sample. Because of the line focusing ability of optical assemblies120, 140 and 160, all of which focus the radiation into substantially asingle line focus, this line focus may be moved laterally back and forthby moving the movable optical assembly 100 and 101 back and forth. Itshould be understood that a wide variety of arrangements of opticalcomponents may exist that can render this feature, of which movableoptical assemblies 100 and 101 are but two examples. These embodimentsand many others are contemplated, which have this capability and aplurality of optical arrangements that accomplish this purpose areencompassed by the appended claims.

FIG. 9 is an illustration showing the lateral horizontal movement of thelight sheet within the biological sample. Beam shaping may also be doneby means of slits or telescopes, independent of the z-direction, toadjust the y-extent of the beam. A telescoping lens 70 shown in FIG. 9may be used to magnify or expand the illumination area, by expanding theline focus 210 in the y-direction. In other words, magnification of theline focus, or rather expanding its lateral extent, may be adjusted bymoving an upstream cylindrical telescoping lens 70 back and forth.

In other embodiments, the magnification may be determined not only byone cylindrical lens, but by a proper optical telescoping lensingsystem. However, the cylindrical lens 70 may have the advantage that itis simple to implement, and spherical errors in y may not impactperformance substantially.

It should be understood that beams using different laser wavelengths maystill be imaged using this optical system because the components are allachromatic. Accordingly, the line foci will still overlap because thefocus is not a property of wavelength. The foci will all still overlapalong a substantial portion of the length of the line, to approximatelythe diffraction limit of the focus. In optical assembly 100, thematerial of lenses may also be chosen (and different) to reducechromatic aberration. Excitation using different color lasers isdiscussed further below with respect to FIG. 20. Detection colors mayalso be selected with the appropriate choice of optical filters in frontof the detector, as discussed below with respect to FIG. 19.

The whole movable optical assembly 100 (or all the elements included inmovable assembly 100) can be moved back and forth along the axis shownin the x-direction, to scan the light sheet through the sample. Thisscanning direction effectively defines the x-axis. Because of the needto move this assembly laterally without altering the focal conditionswithin the sample, the turning mirrors that direct the light into themovable assembly 100 may be configured so as to deliver the light in adirection parallel with the movement direction of movable assembly 100.This situation is illustrated in FIGS. 9 and 10.

It should be understood that any and all optical elements mentionedhere, including the telescoping lens 70, the rotatable structure 51,turning mirrors 61, 62 and 63, and optical assemblies 120, 140 and 160that appear in FIG. 9 on the left hand side of the biological sample 200may also be duplicated and disposed on the right hand side of the sample200. Accordingly, the sample may be illuminated from the left, from theright or from both sides. This system is described below with respect toFIG. 11. The components on the left hand side of light sheet system aredenoted by 100. The components on the right hand side of light sheetsystem are denoted by 100′.

Accordingly, as shown in FIG. 9, the movable optical assembly 100 (or100′) may be moved by a distance Δx. This results in the movement of theline focus 210 by an amount similar or identical to Δx, as shown.

FIG. 10 illustrates a two embodiments 100 of a movable assembly, 100 and101. In the first embodiment 100, similar to movable optical assembly100 of FIG. 9, optical turning elements are included that can enable theentire optical assembly to be moved laterally toward and away from thebiological sample. The effect of this movement of imaging optics is tomove the line focus 210 laterally but in a plane defined by the movementof the optical assembly 100. The movable stage therefore generates thelateral horizontal movement of the light sheet within the biologicalsample for the imaging device.

Embodiment 100 shown in FIG. 10a uses three turning mirrors 61, 62 and63 to deliver three parallel beams into movable assembly 100. The topbeam is redirected by other turning mirrors 74 and 68 and into theoptical subassembly 120. Similarly, the lower beam is redirected by twoother turning mirrors 64 to 66 and into the lower optical assembly 160.The third beam may enter optical subassembly 140 straight on.

In the first embodiment 100 of movable optical assembly shown in FIG.10a , turning mirrors 61, 62 and 63, are used to direct the radiationfrom source 500 into the optical subassemblies 120, 140 and 160. Mirror61 turns the radiation about 90 degrees and into the movable assembly120. Mirror 62 turns the radiation about 90 degrees and into the movableassembly 140. Mirror 63 turns the radiation about 90 degrees and intothe movable assembly 160. With the movable assembly 100, turning mirrors74, 68, 64 and 66, direct the beams into optical subassemblies 120 and160, respectively. For optical subassembly 140, the radiation may enterdirectly from turning mirror 62. Mirrors 74, 68, 64 and 66, may beanalogous or identical to mirrors 53, 54, 56 and 55 in FIG. 5.

In another embodiment 101 of movable optical assembly shown in FIG. 10b, turning mirrors a single turning mirror 61 may be used to direct asingle beam into the moving optical subassembly 101. Mirror 61 turns theradiation about 90 degrees and into the movable assembly 100. Within themovable optical assembly 101, partially reflective/partiallytransmitting mirrors 74, 65, 67 and 69 may be used to direct theradiation from source 500 into the optical subassemblies 120, 140 and160. More specifically, movable assembly then uses turning mirror 74inside movable optical assembly 101 to direct the beam onto thepartially reflecting and partially transmitting mirrors 65, 67 and 69.Each of these partially reflecting and partially transmitting mirrors65, 67 and 69 then direct the radiation into the optical subassemblies120, 140 and 160. Because these subassemblies 120, 140 and 160 aredisposed at angles with respect to the symmetry axis and center opticalsubassembly 140, the angles of the three partially reflecting andpartially transmitting mirrors 64, 65 and 66 are all different, as shownin FIG. 10 b.

As mentioned, the second embodiment of movable optical assembly 101 mayuse the single turning mirror 61 to deflect the radiation from thesource 500 into the movable assembly 101. This embodiment may have fewerturning mirrors than the embodiment shown in FIG. 9, and may thus becheaper or easier to align. However, as with embodiment 100 above,optical turning elements are included that can enable the entire opticalassembly to be moved laterally toward and away from the biologicalsample. The effect of this movement of imaging optics is to move theline focus 210 laterally but in a plane defined by the movement of theoptical assembly 101. The movable stage therefore generates the lateralhorizontal movement of the light sheet within the biological sample forthe imaging device.

FIG. 11 is an illustration of a larger portion of a light sheetmicroscope according to this invention. Illustrated in FIG. 11 is afirst movable optical assembly 100 on the left hand side and a secondmovable optical assembly 100′ on the right hand side. It should beunderstood that this movable optical assembly 100 may be thatillustrated in FIG. 9, or it may be that illustrated in FIG. 10a or 10b, or it may be another embodiment capable of moving the substantiallysingle line focus laterally within the biological sample. Also shown inFIG. 11 is a second movable optical assembly 100′ disposed on theopposite side of the biological sample 200 and cuvette 300. Accordingly,the biological sample may be illuminated from the left by movableoptical assembly 100 and from the right by movable optical assembly100′.

As will be explained in greater detail below, an image may be created bycollecting a plurality of camera images each with the line focus 210 ina different location within the sample. At the beginning of imageacquisition, a partially transmitting turning mirror 57 may directradiation from a source 500 to a mirrored shutter 58, which may redirectthe source radiation 500 into the first movable optical assembly 100.Movable optical assembly 100 may focus the radiation in a substantiallysingle line focus. Then, by moving optical assembly 100 from left toright, the far left edge of the biological sample may first beilluminated, and then the line focus moved successively rightward untilthe line focus reaches the middle of the sample. At this point, ashutter 58 may be moved or retracted to allow the radiation to pass overto the right hand movable optical assembly 100′. The sample may fromthat point onward be illuminated by the right hand movable opticalassembly 100′. After collecting each of these successive images, asingle image may be constructed from these individual scans.

Accordingly, illumination may come from either side to minimize theamount of sample material the light must penetrate. Which of the twosides 100 or 100′ is operative may be selected by a shutter, or a flipmirror 58. Preferably, the shutter or flip mirror 58 is located on thesymmetry axis, as shown, so that the path lengths on the right and leftsides are similar or identical.

It should be understood that right hand movable optical assembly 100′ islargely the mirror image of left hand movable optical assembly 100. Thatis, the angles of the turning mirrors 60′ (in 100′) may be the same asturning mirror 60 but reflected across the symmetry axis shown in FIG.11. The components belonging to mirror image optical assembly 100′ aredesignated by the prime (′) to distinguish them from the left handmovable optical assembly 100. It should be understood that many otherswitching arrangements may be employed to send the radiation from source500 to either the left hand side 100 or the right hand side 100′. Thesealternative optical arrangements are a design choice, and a plurality ofoptical arrangements may exist that accomplish this purpose of routingthe beams in the desired directions. These alternative arrangements fallwithin the scope of the appended claims.

FIG. 12 is an illustration describing the correction for refraction ofthe light beams at the boundary of the transparent liquid vessel 300.The transparent liquid vessel 300 may be glass or quartz, for example.Because of the architecture of the light sheet microscope system asdescribed here, some of the beams of light must necessarily enter thecuvette 300 via the transparent window 360 and 361, with a somewhatoblique angle of incidence β. Accordingly, passing into the cuvette 300,the radiation must pass several boundaries, and so is refracted at adifferent angle than was incident. It should be understood that thelargest refractive effect may occur at the outside air/glass boundary,as this boundary separates materials with the most dissimilar refractiveindices.

As is well known from Snell's law, refraction of the light will thenoccur at the boundary between materials, such that the oblique lightenters the cuvette with a somewhat shallower angle than it enters.Accordingly, because of refraction at glass and liquid boundaries, thefocus of inner, central beam 140 will occur at a different spot inx-direction relative to outer beams 120 and 160. In other words, if theangle between the upper leg and the lower leg is 2β, with which theradiation enters the transparent window 360 and cuvette 300, theradiation may exit with a different angle between the upper and lowerlegs. In general, the exit angle is somewhat smaller than the entranceangle, 2β−. Accordingly, the overlapping line foci will take place at aslightly longer distance D from the transparent window as it would havehad refraction not occurred.

For this reason, it may be advantageous to displace or shift the opticalassembly 100 and 100′ by an amount to accommodate this change in focallength. The amount of the shift D can readily be calculated using basicoptical principles such as Snell's Law. For example, N=1 outsidecuvette, n=1.5 inside. Sin a/sin b=1.5. So if the original angle α₁ is16 degrees, the exit angle may be closer to 11 degrees, and focus mayoccurs at about n×d away from nominal focus. Accordingly, it may beimportant to retard the placement of the movable optical assembly 100 bythis amount, to assure that the line focus falls within the biologicalsample 200 as intended.

Alternatively, the components 120 and 160 may be staggered with respectto component 140 in order to accommodate the change in angle of theirobliquely incident light. Accordingly, it may be possible to advancemiddle optics 140 relative to outer optics 120 and 160 by an amount toaccount for refraction at the material boundaries.

These same operations may be performed on the components in the righthand side optical assembly 100′.

Alternatively, the tilt of the elements 120 and 160 may be adjusted tocompensate for this offset, such that lines overlap perfectly at theline focus 210, or substantially to the diffraction limit, which is tosay within about 5× of the diffraction limit.

In some embodiments, the software running the controller may be toldwhat the index of the transparent window 360 and/or the fluid 1000 is,in order to shift image by an appropriate amount. This may be especiallyimportant in blending of the left hand side and right hand sideilluminated scans.

Because the biological sample 200 may be immersed in a clearing fluid1000, the sample 200 and clearing fluid 1000 may be contained in acuvette 300 as mentioned previously. Since the radiation being must passinto the cuvette, at least a portion of the cuvette may be made from atransparent material such as glass or quartz. The transparent materialwill have a different index of refraction as compared with air. Glassfor example has an index of refraction of about 1.5, as compared to theindex of refraction of air, which is about 1.

Because of the difference in refractive index between the two materials,refraction of the light may occur, resulting in changes to the focalcharacteristics of the light beam. A correction for this effect may bemade, as described above.

The discussion now turns to the computational aspects of the light sheetmicroscope.

Image Formation and Restoration

The discussion now turns to image processing techniques which may beused in combination with the light sheet microscope described above toobtain high resolution, three dimensional images of a biologicalstructure.

FIG. 13 is an illustration showing the pixelated detector which capturesthe image produced by the light sheet microscope from the biologicalsample. FIG. 13 shows the relationship between the line focus 210 andthe detector field of view 310. As shown, the line focus may begenerated at an angle with respect to the optical axis. The detector maybe a pixelated detector such as a CMOS or CCD camera. The figure alsoshows the relationship between the pixelated detector, the line focusand the coordinate system used in the figures to follow.

FIG. 13 shows a line focus generated at an angle with respect to theoptical axis of optical assembly 120 or 160. The y-dimension isgenerally in the direction of the line focus, and along that line. Thex-direction which is the direction of the lateral scanning, isorthogonal to the y-direction. The camera is generally positioned suchthat the sample image falls generally near the center of the field ofview of the detector, in order to efficiently use the pixel area withinthe detector. The optical axis of the objective lens of the imagingmicroscope is aligned along the z-axis and orthogonal to both x- andy-directions.

The data acquired by pixelated detector 600 may include blur,distortion, and/or optical aberrations, and the blur, distortion, and/oroptical aberrations may be a repeatable characteristic of the completeoptical system. The excitation (lasers) may exhibit its own unique andcharacteristic distortions, such that a part of the recorded image'sblur is caused by the intensity distribution of the collimated lasersheets Λ₁ which is not completely flat but a 3D-intensity distribution,see FIG. 15a . Further the x/y-plane defined by the geometry of theexcitation lasers may not be exactly perpendicular to the microscopeturret's z-axis and consequently does not align to the microscope'sfocal plane (the term focal plane means the focal plane of themicroscope on the side of the sample to be magnified), see FIG. 15c forperfect alignment. Further unique and characteristic distortions of therecorded image are caused by the microscope, which exhibits in general afunction Λ₂ that maps the observed 3D-fluorescence distributiontraveling towards the microscope to a 2D image on the surface of thepixelated detector (see FIG. 15d : “microscope image”), whereat thatmapping function Λ₂ may change its shape or orientation across thex/y-plane (as shown in FIG. 15b ) which means that it is spatiallyvarying and as such it rules out a simplistic numerical deconvolution.Finally this 2D microscope image is sampled and converted by thepixelated detector 600 which may come with its own 610 characteristicpoint spread function (PSF) Λ₃.

It should be understood that the term “point spread function” is usedinterchangeably herein with the term “intensity distribution function”and “excitation distribution function”. Each of these terms should beunderstood to refer to a measurable and repeatable noise source withinthe imaging system. Furthermore, the term “imaging system” should beunderstood to may an optical system that can form a two dimensionalimage of an object. Examples of imaging systems are optical lensingsystem that form an image, or a microscope for example.

Knowledge of the functions Λ₁, Λ₂, Λ₃ can be very useful because thesereproducible distortions may be removed from the final raw image Irecorded by the pixelated detector using a numerical 3D restorationprocess resulting in the restored image F, see FIG. 14. This goal may bepursued by employing e.g. an iterative maximum-likelihood optimizationprocess. Alternatively, artificial intelligence driven processes mayeliminate blur, distortion, and/or optical aberrations from recorded araw image I merely on a data driven approach that may not need specificknowledge the of the functions Λ₁, Λ₂, Λ₃. Also a mixture of bothapproaches may be beneficial, especially when the functions Λ₁, Λ₂, Λ₃are known only approximately. As the functions Λ₁, Λ₂, Λ₃ constitute apart of the total system matrix A the system matrix itself may also bedetermined by a maximum likelihood optimization of A parallel to therestoration of the raw image I. A simplistic approach to correct the rawimage I is a deconvolution process, which may be carried out in thefrequency domain. Deconvolutions and the more sophisticated methods fromabove are well known, and the specific details will not be set forthhere. The discussion to follow is directed primarily to the acquisitionof the functions Λ₁, Λ₂, Λ₃.

FIG. 14 shows a schematic illustration of the excitation intensitydistribution Λ₁, the microscope mapping function Λ₂ and the pixelateddetector's PSF Λ₃. The blur, distortion, and/or optical aberrations areshown schematically simply as boxes, to indicate that an incoming signalis altered or transformed in a predictable and reproducible way aseffect of the functions Λ₁, Λ₂, Λ₃. To the extent that Λ₁, Λ₂, Λ₃ can beascertained or measured, as described next, these predictablealterations can be removed from the raw image I by the controller 400 byone of the aforementioned processes. The functions Λ₁, Λ₂, Λ₃ may becalculated numerically by controller 400 based on the data acquiredthrough the lenses and detector. Controller 400 may also perform therestoration process employing these functions Λ₁, Λ₂, Λ₃. The resultwill yield the corrected image F, as shown in FIG. 14. Because Λ₃generally occurs in combination with Λ₂, in some instances only Λ₂, willbe mentioned. However, depending on the context, Λ₂, may be understoodto also include Λ₃.

As shown in FIG. 14, the functions Λ₁, Λ₂, Λ₃ are produced by the lightsheet microscope (laser optics, microscope optics, pixelated detector)and contribute to the data collected and displayed as the raw image Ifrom the biological sample. FIG. 14 illustrates the controller 400 whichmay control image acquisition, image processing, and movement of themovable optical assemblies 100 and 100′.

FIG. 15 portrays the different steps of image formation. FIG. 15a showsa cross section in the x/z plane that shows two iso-intensity lines of alaser sheet. This laser sheet will give rise for fluorescence whenhitting an appropriate sample. The movable optical assemblies 100 and100′ however will produce several light sheets and each of them mayinitially not be oriented perfectly horizontally, but may be tiltedalong the x-axis or y-axis or both. Further, the initial alignment ofthe individual light sheets may be suboptimal. FIG. 15b shows an exampleof the microscope's mapping function Λ₂, here depicted as a series ofdouble-cones whose tails, depicted as the points where the double-conesare joined together, in general define the microscope's focal plane. InFIG. 15b the focal plane really is depicted flat, generally it mayhowever deviate from a plane and exhibit some curvature. The focal planedefines the working distance for sample structures to be imaged with themicroscope's highest resolution. FIG. 15b also shows an example for thespatial variation of the mapping function by different orientations ofthe double-cones. In general, also variations of the cone-shape mayoccur. FIG. 15c shows how the microscope's focal plane and the meanlayer of the light sheet are aligned in order to match the plane ofhighest microscopic resolution with the plane of highest irradiationintensity.

FIG. 15d sketches the image formation employing some pointlike particles750 at different positions that are irradiated by the laser and thatconsequently emit fluorescence light. It is useful to understand themicroscope's imaging process as if it maps the fluorescence lightoriginating from a specific point-like particle 750 in the 3D-samplespace to a blob or to a ring-like area in its 2D-image plane (the termimage plane means the focal plane of the microscope on the side of theimage generated by the microscope). If the particle lies in the focalplane, the sharpest part of the mapping function Λ₂ will give rise to amapped blob, whereas a point-like structure away from the focal plane ofthe microscope will be sampled by the cone-wall-like part of the mappingfunction resulting in a mapped ring in the microscope's image plane. Forpoint-like structures at the side of the optical axis of the microscopethe mapping function may be tilted and consequently a particle outsidethe microscope's focal plane will be mapped to an ellipse. In order togenerate digital images a pixelated detector may be used to detect theimage formed by the microscope on the surface of the pixelated detector.With some simplifications a constant PSF Λ₃ may be attributed to thepixelated detector resulting in some spatially invariant blur in theresulting recorded final raw image I.

Points are not imaged to points because of optical imperfections,scattering and finite resolution (diffraction limit). Furthermore,beyond the line focus of the laser, the laser light diverges and excitesfluorescence in an increasingly broader z-range of the sample, which isimaged by the microscope and introduces additional blur due tosuperimposing adjacent z-layers of the sample to the microscope's imagewhich is recognized as a loss of resolution.

Accordingly, the system may measure point spread function of excitationby moving excitation laterally and vertically. Each of these individualscans may be used to 1) contribute a subset of pixels to the compositetotal raw image I_(C), and 2) be used for computation of the PSF. Thesetwo techniques may be applied to each lateral scan (x-direction), andmay also be applied to each scan taken step-wise through the depth(z-direction) of the sample.

FIG. 16 is a schematic diagram illustrating how an image is assembledfrom a plurality of scans taken with different illumination conditions.Starting to move the movable optical assembly 100 on the left hand side,a scan is collected digitally by a pixelated camera with the line focusin a first position. The line focus is then moved laterally, and anotherscan is taken. The data handling of each of these lateral scans isdiscussed below with respect to FIG. 17. This continues untilapproximately the middle of the sample is reached. At this point, theright hand movable optical assembly is used to illuminate the samplefrom the opposite side. In the middle of the image where theillumination shifts from the left hand side movable optical assembly 100to the right hand side movable optical assembly 100′, the scans areblended electronically using a software algorithm.

This software algorithm may identify noteworthy pixels or sets of pixelsthat provide an identifiable feature in both the right hand scan and theleft hand scan. The scans are then aligned to this point or to a set ofpoints. The scanned images are then blended to assemble what will bereferred to hereafter as the “composite raw Image I_(C)” wherein I_(C)can be understood to be a digital file having the blended dataassociated with each pixel in the pixelated detector. A blending andoffset function may be applied to the data to improve the smoothness andaccuracy of the match. In the middle transition region from left sideoptical assembly 100 to right side optical assembly 100′, thecontribution from the side which has been active up until the transitionmay be given a gradually reduced weight, whereas the contribution fromthe side coming on line is given a gradually increasing weight. Thistransition and the functions used are shown in FIG. 16.

FIG. 17 is an illustration showing the step-wise lateral horizontalscanning of the light sheet through the biological sample. For eachimage, 1, 2, 3, 4 and 5, the line focus 210 is shifted laterally bytranslating the movable optical assembly 100. An image is taken with theline focus as shown in each image. Although five line foci are shown inFIG. 17, it should be understood that there may be many more images andmany more line focus positions than just five. The lateral separation(i.e. the distance between the scans) may be a performance choice thatcan be made, based on the numerical aperture selection made as describedabove with respect to FIGS. 8 and 9.

The detector and computer may capture a sequence of images 1-5 as shownin FIG. 17. These raw images I may be used as input for imagerestoration and may also be used to calculate the functions Λ₁ and Λ₂.On the other hand, they may be used as input for assembly of thecomposite raw image I_(C) that is composed of not image-restored stripesof raw image I sequences. As the image restoration process takes intoaccount, if available, all sequences of scanned line foci andz-positions (scanned through the depth of the sample), the assembly ofcomposite restored images F_(C) may be performed by the restorationprocess itself. These techniques are described below.

Firstly, the portions of the image which include the line focus 210which is depicted as the cross-hatched portions, may be assembled stripby strip into the final composite raw image I_(C) shown at the bottom ofFIG. 17. The composite raw image may therefore have subsets of each ofscans 1-5 included therein. That portion of the scans 1-5 may beselected whose width is approximately within the Rayleigh length z_(R)(see FIG. 16) of the beam waist. Data outside that range may be ignoredin the formation of the composite image I_(C), however this date maystill contribute to the calculation of the functions Λ₁ and Λ₂, asdescribed below, or for the image restoration process. Accordingly, theRayleigh length may determine the step size of the sequential scans. Theblending of the transitions may be handled as was the left/righttransition shown in FIG. 16. Performing the blending algorithm overthese scanned areas may result in superior homogeneity. It may beimportant to avoid adjusting any parameters during any scan, such thatpixels can be compared and areas matched.

The width of strip along x (i.e. the width of the illumination) maydepend on the tightness of focus w₀ (see FIG. 16) and thus on the NA ofthe optical system. By increasing resolution (increasing NA and thusincreasing the tightness of focus wo) one narrows the strip anddecreases the number of useful pixels for the composite image. Typicalis 10 steps per image with about 200 pixels per step, resulting inusually 2160 useful pixels in x-direction (scanning direction). AR is1/10 or 1/15 or so. If the aspect ratio 10, there may be only 200 usefulpixels/image, for example.

In some embodiments, it is advantageous to use a relatively largenumerical aperture and perform a large number of steps. In otherembodiments, a larger Rayleigh length z_(R) and fewer steps are moreappropriate. Such details will depend on the application and the typeand quantity of data being sought.

Accordingly, the composite raw image I_(C) may be assembled from theindividual scans 1-5. As with the center blending algorithm thecomposited image may be assembled by identifying noteworthy features ina scan sequence, and fitting the data based on the characteristics ofthe noteworthy feature. Using this method, a composite raw image I_(C)may be reconstructed from a series of sequential, overlapping lightsheet scans. However, each of scans 1-5 may also be used in thecomputation of the functions Λ₁ and Λ₂, as described below, or for theimage restoration process.

However, in addition, the entirety of the data from each of scan 1-5 mayalso be fed to the computer or controller, because these multiple imagesof the same field of view contain information on the functions Λ₁ andΛ₂, namely the intensity distribution of the laser and the mappingfunction of the microscope. In other words, as the line focus 210 isswept over a field of view, different portions of the Gaussian beamilluminate different portions of the sample. Accordingly andimportantly, ALL the data collected in EVERY scan may contribute to thecalculation of the function Λ₂. This data may then be used for therestoration process that forms from the raw images I the restored imagesF.

In other words, each of the images 1-5 may contain, both within thecross hatched regions and elsewhere, information about the functions Λ₁and Λ₂. This includes portions that are not being significantlyilluminated by the line focus 210. Accordingly, the function Λ₂ may havea contributions from the entire first image I₁₁. It may have anothercontribution from the entire second image 112. It may have yet anothercontribution from the entire third image 113 and so on. The totalfunction Λ₁ that covers the complete spatial extent of the applied laserintensity distribution from the far left part of the line focus to thefar right part of the line focus, and the total function Λ₂ of themicroscope mapping functions over the complete spatial extent of themicroscope's FOV may be derived from all the individual scans. Thistechnique is then also applied to scans done through the depth of thesample, as described next.

FIG. 18 is an illustration showing scanning in the lateral dimension(x-dimension, FIG. 18a ) and scanning in depth (z-dimension, FIG. 18b )to produce a three dimensional image by the light sheet microscope fromthe biological sample. The x-dimension scanning may be produced bymoving the movable optical assembly 100 and 100′ laterally in thex-dimension, which has the effect of moving the line focus 210 laterallyacross the biological sample 200. The separation between successivescans may be di Furthermore, di may be related to the beam waist andthus to numerical aperture and Rayleigh length z_(R) of the light sheet.Similarly, the width of the line focus d₂ may also be related to thebeam waist and thus to numerical aperture and Rayleigh length z_(R) ofthe light sheet.

The sample may then be scanned through its depth in the z-direction,orthogonal to the focal plane of the microscope. The depth scanningshown in FIG. 18b may be accomplished by moving the biological sample upand down in the z-dimension, and then repeating the lateral scanningjust described.

As was done with the lateral (x-direction) scans, the composite rawthree dimensional image I_(C) may have subsets of each of the depthscans included therein. That portion of the scans may be selected whosewidth is approximately within the Rayleigh length of the beam waist. Theblending of the transitions may be handled as was done with theleft/right transition shown in FIG. 16. Performing the blendingalgorithm over these scanned areas may result in superior homogeneity.Accordingly, all the data from all the scans is used either in thegeneration of the composite image I_(C). Alternatively all the data fromall the scans, specifically of the depth-scans could be used for thecalculation of the z-variation of the functions Λ₁ and Λ₂, namely thez-variation of the intensity distribution of the laser and the mappingfunction of the microscope; having accomplished the measurement thefunctions Λ₁, Λ₂ altogether with Λ₃, all the data from all the scans maybe used as input for the restoration process.

To give an example, an acceptable acquisition speed and resolution mayrequire on the order of 20 individual scans. If B is the total width ofthe image plane, as shown in FIG. 18, then d₁=B/N, n=1 to N, typicallyless than 20 individual scans n. N can be as much as 100 but typicallyless than 20. sheets should ideally overlap. d₁ should be chosen keepingin mind the size of the structures being imaged.

In some embodiments, the same plane may be addressed several times. Therest of the sample does not experience sharp focused light. If one usesa sampling which is too high, photo bleaching may get relevant becausethe energy transfer of excitation lasers light to the sample grows withsmaller sampling steps and tighter laser focus. If one uses a very lownumber of samples, it becomes tantamount to using a confocal microscope.

If a standard confocal microscope is used to address all layers, thewhole sample is illuminated during image acquisition. As a result, thewhole sample is bleached repeatedly during scanning, generatingdestructive heat as well. Using the scanning method of the presentedinvention however, the sample is illuminated by the laser specificallyonly in the plane of interest which may be moved vertically to the nextplane of interest.

When an image is collected using several scans with the line focusmoving between successive scans, the line foci may be overlapping. Thismay give improved performance and accuracy, because identifiablefeatures within the image can be used to align the scans accuratelybefore merging them into the final image.

Finally, one can combine all the data to obtain an overall descriptionof the optical degradation processes by the functions Λ₁ and Λ₂, namelythe z-variation of the intensity distribution of the laser and themapping function of the microscope. It should be noted that each of thethree beams in the movable optical assemblies 120, 140 and 160 exhibitsa similar function Λ₁ because they have similar optical properties, butit may be noteworthy to point out the benefit when the geometricorientation of the three movable optical assemblies 120, 140 and 160 ismeticulously aligned.

The restoration process may yield better a signal-to-noise ratio (SNR)and fewer blurring and deformation artifacts because the measured inputto the restoration addresses each point in the sample volume withdifferent excitations in x and z. Accordingly, the plurality of scanscan be seen as a system of coupled linear transformations of the trueunblurred and undeformed 3D-representation of the sample that can beused to set up an objective function that reaches its minimum for thistrue 3D-representation of the sample—which describes the usual approachof minimizing an objective function for getting a restorated image of asample.

For a measurement with a specific sample one may separate the effect onthe optical degradation imposed by the functions Λ₁ and Λ₂, namely theintensity distribution of the lasers and the mapping function of themicroscope, between the part that originates from the optical systemonly and the part that originates due to the influence of the currentsample. In order to do so, it is beneficial to perform measurements withideal sample structures as for example glass beads (nano beads) that areembedded in a preferentially mechanically stable environment of similarrefractive index as in the measurement of the specific sample, as forexample agarose. As these measurements are performed on point-likeobjects in a surrounding environment of constant refractive index, theexcitation light may be regarded as travelling through the probe withoutbeing deformed. As such the functions Λ₁ and Λ₂ resulting out of thesemeasurements plus the function Λ₃ (describing the PSF of the pixelateddetector) represent altogether the characteristics of the image formingprocess of the laser microscope, however without additional degradationsinduced by the biological sample.

The influence of the sample itself may deviate the exciting lasers fromtheir primary paths whilst travelling deeper and deeper into the sample.Consequently portions of the sample may get illuminated that would stayunexposed if the lasers have travelled without being interacting withthe sample. This deviation of function Λ₁ may be disentangled forexample by techniques that compare exposures coming from focal linepositions of the left hand movable optical assembly 100 and the righthand movable optical assembly 100′ that both should have exposed thesame part of the sample, given that there is no influence and deviationby the sample itself. Concerning Λ₁, this deviation may not only be ageometric deformation of the laser intensity distribution but also anoverall attenuation and also so called shadows caused by an attenuationof certain sample substructures that appear opaque to the laser lightdue to insufficient or impossible probe clearing.

The influence of the sample itself may change also the microscope'smapping function Λ₂ and this deviation in general will change whilstobserving z-planes of the sample that lie, from the microscope'sperspective, deeper and deeper inside the sample. This is a consequenceof the increasing path length that the emission light has to travelthrough the sample towards the microscope. Consequently the mappingfunction Λ₂ may be distorted in several ways; firstly the geometry ofthe mapping, depicted for example as double-cones in FIG. 15 may bedeformed in a way specific to the sample. Secondly, the microscope'sfocal plane may be distorted and a curvature may be introduced, whichmeans that the positions in x/y/z where the local mapping function hasits tail, depicted as the points where the double-cones in FIG. 15 arejoined together, may change. This deviation of function Λ₂ may,alongside with for example artificial intelligence based approaches, becalculated during the restorations process itself.

A way to calculate the sample-induced deviation of function Λ₂, (andalso the deviation of function Λ₂) may be performed by a semi-blindrestoration process that starts to restore the image of the sample byinitially using the undisturbed functions Λ₁ and Λ₂, (see previousparagraphs) for an explanation of how these undistorted functions Λ₁ andΛ₂ could be measured and calculated. After some iterations, given thenumerical method operates iteratively, the functions Λ₁ and Λ₂ may bethemselves be optimized in a way that minimizes another objectivefunction where the currently achieved image of the sample enters as aconstant input entity. From this stage on, both the image of the sampleand the sample distorted functions Δ₁ and Δ₂ may by optimized by analternating approach. It should be noted that this description is oneamong others that can be used to optimize both the image of sample andthe sample distorted functions Λ₁ and Λ₂. In yet another alternative,totally blind restoration may be performed without measuring anyfunctions Λ₁, Λ₂ and Λ₃ at all. However this is very time consuming andmay not deliver a meaningful result at all, as it is in general anon-convex optimization problem that may end up in a local minimumrather than the global minimum. Therefore also in blind restorationprocesses one usually makes some appropriate assumptions of how thefunctions Λ₁, Λ₂ and Λ₃ may be structured. If these assumptions arederived from measurements and calculation of undisturbed functions Λ₁and Λ₂ according to previous paragraphs (a starting assumption forfunction Λ₃ of the pixelated detector may also be given), one ends upfor example in the semi-blind restoration process described above.

The discussion now turns to the mechanical and optical aspects of thelight sheet microscope.

Optical Sources and Alternative Optical Paths

FIG. 19 is an illustration showing additional optical components whichmay be advantageous for the functioning of the light sheet microscopesystem. The optical path may include at least two wavelength or colorselectors. First wavelength selector may be a filter wheel 550 on thesource of the radiation 500. The second wavelength selector may beanother filter wheel 660. on the detection path. The first filter wheel550 may select or choose the working wavelength for the light sheetmicroscope excitation and detection. Ordinarily detection isperpendicular (along vertical axis) to excitation but not necessarily.

A filter wheel may be an assembly of apertures arranged in a circle andcovered by a material that filters some wavelengths of light whileallowing other wavelengths to pass. Different apertures may be coveredby different materials, and therefor transmit different wavelengths.Accordingly, the transmitted wavelength may be selected by rotating thefilter wheel, to place different apertures in the beam path. One filterwheel 550 may be used after the source 500 and another filter wheel 660may be used in front of the detector 600.

FIG. 20 is an illustration showing some exemplary optical sources forthe light sheet microscope excitation and detection. In one embodiment,a white light source 500 (FIG. 20a ) can be used, and filter wheel 550that then selects, for example, any of eight or so constituentwavelengths. Filter wheel 560 may have 8 colored filters, and may beused to serially select which wavelength to view by rotating filterwheel 550. The filter wheel 550 may have settings capable of passingimportant radiation lines, well known in fluorescence microscopy. Theselines may include the six excitation lines at 488, 515, 553, 591, 640,785 nm, for example.

In other embodiments (FIG. 20b ), separate light sources may be used,for example laser light source 521, laser light source 522, laser lightsource 523, laser light source 524, and laser light source 525. Eachlight source may have an associated turning mirror which deflects theemitted radiation into an imaging lens 530 which may collimate or focusthe radiation and input the radiation into an optical fiber 510. Theoptical fiber 510 may transmit the collimated radiation to anothercomponent 540 which may further modify the radiation and finally into acollimating lens 580. From collimating lens 580, the radiation mayfollow the path shown in FIG. 19. In other words, in the embodimentshown in FIG. 20, the single source 500 may be substituted with aplurality of discrete light sources emitting at different wavelengths521-525. There may be 5 lasers as shown in FIG. 20. The five lasersources may be coupled by dichroic elements as shown. The laser outputsmay all then be focused by a lens 530 into a fiber optic cable 510. Thediverging fiber optic output may then be collimated by collimating lens580. Of course, there may be fewer or more discrete light sources otherthan those shown as 521-525.

Alternatively, the light source may use a laser combiner or asupercontinuous laser emitting in a very broad band 450-2000 nm. Thisrange may cover four or more excitation lines. The laser combiner may besoftware controlled to add continuously varying quantity of any colorlaser.

In operation, each of the five lasers may be switched on and off. Anacousto-optical modulator may be convenient for this switching. The fivelasers may be high speed, femtosecond pulse emitters, so as to capturevery short events.

FIG. 21 is an illustration showing the components of a detection pathfor the light sheet microscope. The detector may include an imagingobjective lens 620 and another filter wheel 660. Like filter wheel 550,filter wheel 660 may only pass certain wavelengths of light. Forexample, it may be desirable to image all structures which are emittingat 515 nm for example. In this situation, the filter wheel 660 may berotated until the filter element that passes 515 is placed into the pathof the radiation. The selected wavelength may then be imaged onto thepixelated detector 600 by a number of other optical components includinga microscope objective lens 670, depending on the application. Finally,the pixelated detector 600 may measure the light intensity at theselected wavelength and in the particular pixel element. As shown inFIG. 21, there may also be a magnifying/demagnifying element 59, whichchanges the magnification of the imaged sample without changing theobjective lens 670. It should be understood that pixelated detector 600depicted in FIG. 21 may be a similar or identical to pixelated detector600 depicted in FIG. 19 and referred to in FIG. 14.

Alternatively, the detector may use two or more cameras which respond todifferent colors. The detector may alternatively use a color camera.

Independent Motions

One of the major advantages of the light sheet microscope describedhere, is that the components can be moved independently of one another.For example the line foci may always fall in a single plane defined bythe motion of 100 and placement of its components 120, 140 and 160. Thisconfiguration of parts defines the image plane in which the sample isplaced, and the focal plane defined by the line foci. This plane mayremain fixed while many other components are moved with respect to thisplane. Accordingly, once the plane is established and detection isfocused or adjusted with respect to it, these adjustments may not needto be made again. This may be true even if the sample is changed, or theimaging lens is changed, or the clearing fluid is changed. The movablesample stage may be configured to always hold the sample in this plane.These advantageous features may be accomplished by using stages withindependently movable stages with respect to the image plane andarranged as described next.

FIGS. 22 and 22 a, which includes FIGS. 22a, 22b, and 22c , areillustrations showing a first important mechanical/optical aspect of thelight sheet microscope. A turret 10 may be used which holds a pluralityof objective lenses. The optical axis of each of these lenses may beparallel. Using the design shown in FIG. 22, the turret 10 may berotated and a different objective lens may be selected, without movingthe sample 200 or any of the excitation or other detection optics. Therotation may be accomplished without moving the sample or the focalplane of the line focuses. This may be important for obtaining reliable,reproducible images at a varying magnification. The movement isaccommodated by having the cuvette 300 shaped with recesses that allowthe objective lenses to penetrate the plane of the biological sample,even when these lenses are not in use. This allows the imaging ofdifferent areas, with different magnifications, in a manner that isexceedingly easy and fast.

For example, a microscope is described which has the three objectivelenses 1, 2 and 3 mounted on a rotating turret 10. The optical axes ofthe lenses 1, 2 and 3 may be parallel, as mounted in the turret 10. Oneof the plurality of lenses, say lens number 3, may be the imaging lenswhich is actually in use. The others 1 and 2 of the plurality allowdifferent magnifications and fields of view to be chosen, but are not inuse at the present time. When an image is taken, the operative lens 3 islowered into position just above the biological sample, such that thelens is submerged in the clearing fluid 1000 and contained within theperimeter of the cuvette 300. Since the non-operative lenses 1 and 2 aremounted also to the turret 10 and thus coupled to the same mechanism,these lenses 1 and 2 are lowered as well. Two cutouts or relieved areasor curved surfaces or voids 350 and 351 are designed into the cuvette toallow these lenses 1 and 2 to be lowered as well, but not to interferewith any other structures in the microscope. Accordingly, when theoperative lens is submerged, the at least one inoperative lens is notsubmerged (i.e. is located beside the container)

The other cut outs 950 and 951 may be made in the movable stage 900which supports the movable optical assembly 100 or 101. Theses cutoutsmay also be dimensioned to admit any one of the lenses 1, 2 or 3.Together, cutout 352 and 952 may admit lens 2 for example, while cutout351 along with another cutout in stage 900 (not shown for convenience ofrendering) may admit lens 1. The movable sample stage 800 may be movablyplaced within the cuvette 300 as shown.

When a different magnification or field of view is desired, the turret10 may be raised until it clears the cuvette 300 and the turret 10 isthen rotated to select a different operative lens. With the new lens inposition, the turret is again lowered into the cuvette 300 to a positionjust above the sample. Because of the relieved areas 350, 351 for lenses1 and 2, these lenses can be lowered as well without mechanicalinterference, because of the cutouts or relieved areas or curvedsurfaces or voids 350 and 351.

FIG. 22a is a plan view illustration showing the ability to rotate aturret holding a plurality of objective lenses. FIG. 22b is aperspective illustration of the sample stage 800 and cuvette 300,accommodating the turreted lenses 1, 2 and 3. FIG. 22c is a perspectiveillustration of the turreted lenses 1, 2 and 3. The turret 10 is shownschematically in FIG. 22c , as a rotatable mechanism to which threelenses, 1, 2 and 3 are attached. As shown in FIG. 22c , the differentlenses may have different physical dimensions. In particular, one of thelenses 1, 2 or 3 may be substantially longer than the other two. One mayalso be wider in diameter than the others. The cutouts or relieved areas350 in the cuvette 300 may be shaped so as to accommodate thisdimensions. Cutout 350 may be circular and may admit the operative lens3. Cut outs 351 and 352 may be portions of a circle, and shaped to admitthe otherwise interfering portion of lenses 1, 2 or 3 as was shown inFIG. 22a . Accordingly, lens selection can be changed without movinganything but the lens carriage. i.e., without moving sample 200, cuvette300 or light sheet plane or line focus plane 210. As mentioned, theother cut outs 950 and 951 may be made in the movable stage 900 whichsupports the movable optical assembly 100 or 101. Theses cutouts mayalso be dimensioned to admit any one of the lenses 1, 2 or 3. Together,cutout 352 and 952 may admit lens 2 for example, while cutout 351 alongwith another cutout in stage 900 (not shown for convenience ofrendering) may admit lens 1. The movable sample stage 800 may be movablyplaced within the cuvette 300 as shown.

FIG. 23 is another plan view illustration showing how the light sheet orline focal plane 210 may be moved laterally independently of theturreted objective microscope. FIG. 23 is similar to FIG. 22 except thatthe movable optical assemblies 100 and 100′ are also shown in relationto the cuvette 300. The sample holder 800 is also shown in FIG. 23 andits motion is also shown diagrammatically. Cut outs 351, 352, 951 and952 described above are also illustrated in FIG. 23, as are cuvette 300and sample stage 800.

It should be understood that although the scanning direction, that is,the direction of lateral motion of the line focus, is described as beinggenerally in the x-direction, and therefore orthogonal to the extent ofthe line focus, the scanning could also be performed in the y-direction(i.e. parallel to the extent of the line focus).

The software may know the focal distance of each lens so may focusautomatically. The focus may be obtained simply by moving theoperational objective up and down in the z-dimension. This is possiblebecause excitation plane and the sample have not moved during thisprocess. This capability offers substantial ease of use for changingsamples and ease of use changing lens/imaging. It also lends itself torobotic, or automated functioning.

As shown in FIG. 23, the movable optical assemblies 100 and 100′ may bemoved laterally without moving or interfering with any other features ofthe light sheet microscope. The turreted lenses 1, 2 and 3 on turret 10may also be moved without affecting or disturbing any other feature ofthe light sheet microscope. As described previously, cutouts 350, 351,352, 951 and 952 may serve to admit each and any of objective lenses 1,2 or 3. Lastly, the motion of the sample holder 800 may also be movedindependently, and which movement is described further below withrespect to FIG. 24.

Accordingly, for a turret with three objective lenses, the lenses areall allowed to dip into the plane of the sample. The lens may be raisedby a lens raising mechanism, a new viewing lens rotated into position,and finally lowered into the fluid. Accordingly, the motion may beraise, then rotate, then lower. The system therefore fulfils the objectto keep the lenses within setup while they are changed, and withoutmoving any other components. The turret 10 holds the optical axes of thelenses in parallel and on its circular, rotating tray.

The light sheet microscope system may have yet other independentlymovable features. It should be understood that the openings 350, 351,352, 951, 952, that admit the turreted objective lenses may be made inthe movable optical assemblies 100, 100″ or they may be made in othersolid surfaces of the microscope body, such as optical platforms andstages. But in any case, theses openings 350, 950 and 951 are formed inthe solid material of the light sheet microscope in order to allow themovements just described.

The light sheet microscope system may have yet other independentlymovable systems. The sample stage 800 and cuvette 300 may also bemovable independently from the other systems. Throughout these motions,the light sheet image plane 210 may remain fixed. This configuration maybe ideal for robotic handling and trays. These capabilities areillustrated in FIGS. 24, 25 and 26.

FIG. 24 illustrates another independently movable feature of the lightsheet microscope. The cuvette stage 930 which supports the cuvette 300with clearing solution 1000 may also be raised and lowered independentlyof the movable optical assemblies 100 and 100′ the objective lenses 1, 2and 3 on turret 10, and the sample 200 on sample holder 800. Thiscapability is illustrated in FIG. 24a and FIG. 24 b.

In FIG. 24a , the operative lens 700 is in position just above thebiological sample 200. In this position, the operative lens 700 issubmerged in the clearing fluid 1000. If the clearing solution of thesample 200 needs to be manipulated or changed, the stage 930 supportingthe cuvette 300 may be lowered. This lowering may elevate or withdrawthe biological sample 200 from the clearing fluid 1000. As shown in FIG.24b , either the sample 200 or the clearing fluid 1000 may be changed ormanipulated without changing or interfering with any other aspect of thelight sheet microscope. The cuvette can be lowered without affecting thesample or sample holder. This implies that the user can take sample outwithout dipping into solution. Focusing, e.g., may still be adjustedeven though sample has changed. The light sheet plane and detector maystay in the same relative position. No other operations are needed tomove the liquid filled vessel out of the way, or to return it intoposition with fresh fluid, submerging the sample for imaging. As before,transparent windows 360 and 361 may admit the radiation to the sample200.

FIGS. 25a and 25b show further details of the sample holder 800, whichalso has some important features. The sample holder 800 may include asample stage arm 810 which may extend laterally from a supporting point820. This supporting point 820 may be coupled to a movable actuator (notshown) that can raise and lower the supporting point 820. By moving thesupporting point, the sample 200 may be raised or lowered into or out ofthe clearing solutions. The side view of FIG. 25b illustrated therelationship of the movable stage 930, sample stage 800, and cuvette300. The sample stage 800 coupled to a sample supporting point 820. Thissupporting point 820 may be moved laterally or vertically by athumbscrew or rack and pinion, for example.

It should be understood that if the sample 200 is raised, then theoperative objective lens may also need to be raised to allow clearance.However, with movable supporting point 820, the sample may be removedfrom the clearing fluid and manipulated or exchanged without disturbingany other aspects of the light sheet microscope.

The two independent motions, of the cuvette stage 930, and the samplesupporting point 820 are shown in FIG. 25b . FIG. 25a shows thecomponents in their operative orientations in plan view. The cuvette 300may have a large circular hole 350 in the top surface 350 that willadmit the operational objective lens. This aperture 350 may allow thelens to be lowered into the clearing fluid 1000 until it is the properdistance from the biological sample such that the structures are infocus. The operational lens may then be raised until it clears thecuvette 300, and the lens assembly and turret may be rotated to placeanother objective lens into the operational position. The othernon-operational lenses will be lowered into the cutaway surfaces 351,352, 951 and 952.

As shown in FIG. 25, the biological sample 200 may be mounted on amovable sample stage 800. The sample stage 800 may be a motorized x, y,z stage, movable in three dimensions.

Thus, the sample stage may be movable vertically with a throw of, forexample, about 30 mm. However the sample stage may also be movablelaterally, with a throw of about 80 mm. Thus, the sample can be movedlaterally with respect to the movable optical assemblies butimportantly, it may also be movable vertically. The vertical movementmay enable the three dimensional imaging capability discussed earlier.The lateral scanning, as explained before, is accomplished by moving themovable optical assemblies 100 and 100′ laterally with respect to thesample, rather than by moving the sample laterally. Accordingly theplane of the line focus, that is, the plane within which the line focusmoves, is established by the motion of the movable optical assemblies100 and 100′ and may not change during operation in general.

The movable sample holder 800 allows for imaging large samples e.g. upto 100×100 mm. Accordingly, the field of view/magnification mayaccommodate a detector with an active area on the order of 22 mm.

Accordingly, the sample holder may be manipulated independently ofoptics, fluidics, excitation and detection. The sample may be movedvertically in the plane for different depths and laterally for a changedimaging location. The ability to change samples without touching theexcitation or detection optics offers significant benefits in terms ofease of use. The fluid receptacle or cuvette 300 can move vertically inthe z-direction independently of the sample, the detector and theexcitation. Accordingly, it may be possible to change samples withouttouching the optical system. It is also possible to change samples 200without touching the sample holder 800.

It should also be understood that artificial intelligence techniques,such as machine learning and deep learning, using for example tensorflow records, may be used to improve the final image quality.

FIG. 26a is a side view illustration of the structures shown in planview of FIG. 25. FIG. 26a shown the sample 200 submerged in the cuvette300 and clearing fluid 1000. FIG. 26b shows the sample 200 retractedfrom the cuvette 300 and clearing fluid 1000. The light sheet plane 210may stay in position relative to everything but the cuvette 300.

FIG. 27 is an illustration showing how multiple samples may be handledusing the novel sample holder. Because of the large throw (25 mm in thislateral dimension), multiple biological samples may be loaded on thesample holder simultaneously. In FIG. 27, five samples 810 are shownloaded on to the sample holder 800. Using the lateral actuator, each ofthese samples can successively be brought in to the plane of the linefoci and imaged. Accordingly, Multiple biological samples may be imagedwithout moving any other structure with in the light sheet microscope.No optics, no detector, no fluidics, no imaging systems need to bemoved. As a result, the conditions under which successive images aretaken are as nearly as is possible, identical. This allows forparticularly easy and straightforward automated or robotic operation.

The light sheet microscope may be made by machining and anodizingaluminum, using a combination of publicly available lenses, lasers,optical elements such as turning mirrors, movable stages, stepper andcontinuous motors, for example, in addition to custom parts. Thedetector may be a CCD camera readily available from a variety ofsources. The device may be calibrated and focused using standardprocedures in microscopy, and by imaging materials with knownattributes, such as glass beads. The images may be displayed on monitorson the machine or remotely over the internet, for example.

Among all the afore described independent motions, the light sheet focalplane always stays in the same position however, within the cuvette andwith respect to the system. The detection locks to this excitation focalplane.

Accordingly, a light sheet microscope is described. The microscope mayinclude a detector which forms an image of the biological sample throughimaging optics, wherein the biological sample disposed in a focal planeof the imaging optics, and a container holding a quantity of fluid anddisposed on a movable first stage, movable in the z-direction, whereinthe z-direction is orthogonal to the focal plane. The microscope mayalso include a sample holder holding the biological sample, wherein thebiological sample is immersed in the fluid and the biological sample isin the focal plane. The first stage may have a range of motion such thatthe sample can be both immersed in the fluid and in the focal plane andthen withdrawn from the fluid by the motion of the first stage, whereinthe first stage moves the container independently of the sample holder,the imaging optics and the detector.

The light sheet microscope may further comprise a movable opticalsubassembly configured to focus a plurality of light beams into a singlestraight line that defines a light sheet, and wherein the light sheetlies in the focal plane of the imaging optics and therefore also in thebiological sample. The light sheet microscope may also include a movablesecond stage that holds the movable optical subassembly. The secondmovable stage may move laterally and parallel to the focal plane, andperpendicularly to the z-direction, thereby moving the light sheet to adifferent laterally adjacent region of the biological sample,

The plurality of light beams may comprise at least three light beams,each of which is separated by an angle, wherein the at least three lightbeams are focused by the movable optical subassembly all to the samestraight line defining the light sheet, and wherein the light sheet liesin the focal plane. The plurality of light beams may further comprisesix light beams, with three of the six light beams on either of twoadjacent sides of the biological sample and wherein each of the threelight beams is separated by an angle, and with each set of three beamsfocused to a single straight line by two movable optical subassembliesdisposed on either side of the biological sample. Within the light sheetmicroscope, the container, the sample holder, the biological sample, andfirst and second stages may all be movable without changing the focalplane of the imaging optics and a location of the light sheet.

The different laterally adjacent regions may comprise at least 5 regionsspaced laterally adjacent within the biological sample but within thefocal plane of the imaging optics. Furthermore, by moving the containerholding the fluid in the z-direction, the sample holder and biologicalsample may be held aloft of the container and fluid, such that thebiological ample can be manipulated without affecting any one of thefocal plane, the detector, the imaging optics, the light sheet ortouching the fluid. The sample holder may be moved without changing arelative orientation of the imaging optics or the focal plane or thelight sheet. The sample holder may be configured to allow the biologicalsample to be manipulated without changing a relative orientation of theimaging optics or the focal plane or the light sheet.

The light sheet microscope may further comprise an imaging lensstructure upon which the imaging optics is mounted, wherein the imaginglens structure is also movable in the z-direction and wherein theimaging optics are configured to form the image on the detector, whereinthe lens structure, the container, the sample holder, the biologicalsample, and first and second stages are all movable without changing thefocal plane.

Within the light sheet microscope, the detector may comprise a pixelateddetector wherein the imaging optics form a pixelated image of the focalplane on the pixelated detector, and wherein the imaging optics may bemoved without moving the pixelated detector, the biological sample orthe container. The imaging optics may be submerged in the fluid bymoving the lens structure downward in the z-direction on the movablesecond stage or by moving the first stage and container upward in thez-direction on the movable first stage, and wherein the fluid is aclearing fluid that renders the biological sample more transparent toradiation. The sample can be removed and replaced by another samplewithout moving the focal plane or the lens structure. At least a portionof the imaging optics may be submerged in the fluid above the sample andsample stage to image the irradiated sample.

The light sheet microscope may further comprise a movable third stagecoupled to the sample holder, configured to move the biological samplein the sample holder in the z-direction to illuminate multiple depths inthe biological sample with the plurality of light beams. The light sheetmay move in the z-direction to irradiate multiple areas at differentdepths within the sample by moving the third stage in the z-direction,without affecting the position of the first stage or second stage.

The different depths may comprise at least 5 different depths. Thesample holder on the movable third stage, the lens structure on themovable first stage, and the container on the movable second stage canall move independently of, and without disturbing, the focal plane. Themovable third stage may have a throw of about 20-40 mm in thez-direction, and 70-90 mm laterally, orthogonal to the z-direction, andthe movable first stage may have a throw of about 40-50 mm in thez-direction.

The biological sample may be replaced with a new biological sample,without disturbing the sample stage, the lens structure, or the focalplane, by moving the container on the movable first stage to expose thebiological sample.

While various details have been described in conjunction with theexemplary implementations outlined above, various alternatives,modifications, variations, improvements, and/or substantial equivalents,whether known or that are or may be presently unforeseen, may becomeapparent upon reviewing the foregoing disclosure. Accordingly, theexemplary implementations set forth above, are intended to beillustrative, not limiting.

1. A light sheet microscope for imaging a biological sample, located ona sample holder, comprising: a detector which forms an image of thebiological sample through imaging optics, wherein the biological sampledisposed in a focal plane of the imaging optics; a container holding aquantity of fluid and disposed on a movable first stage, movable in thez-direction, wherein the z-direction is orthogonal to the focal plane; asample holder holding the biological sample, wherein the biologicalsample is immersed in the fluid and the biological sample is in thefocal plane; wherein the first stage has a range of motion such that thesample can be both immersed in the fluid and in the focal plane and thenwithdrawn from the fluid by the motion of the first stage, wherein thefirst stage moves the container independently of the sample holder, theimaging optics and the detector.
 2. The light sheet microscope of claim1, further comprising: a movable optical subassembly configured to focusa plurality of light beams into a single straight line that defines alight sheet, and wherein the light sheet lies in the focal plane of theimaging optics and therefore also in the biological sample; and amovable second stage that holds the movable optical subassembly, whereinthe second movable stage moves laterally and parallel to the focalplane, and perpendicularly to the z-direction, thereby moving the lightsheet to a different laterally adjacent region of the biological sample,3. The light sheet microscope as in claim 2, wherein the plurality oflight beams comprises at least three light beams, each of which isseparated by an angle, wherein the at least three light beams arefocused by the movable optical subassembly all to the same straight linedefining the light sheet, and wherein the light sheet lies in the focalplane.
 4. The light sheet microscope as in claim 2, wherein theplurality of light beams comprises six light beams, with three of thesix light beams on either of two adjacent sides of the biological sampleand wherein each of the three light beams is separated by an angle, andwith each set of three beams focused to a single straight line by twomovable optical subassemblies disposed on either side of the biologicalsample.
 5. The light sheet microscope as in claim 2, wherein thecontainer, the sample holder, the biological sample, and first andsecond stages are all movable without changing the focal plane of theimaging optics and a location of the light sheet.
 6. The light sheetmicroscope as in claim 2, wherein the different laterally adjacentregion comprises at least 5 regions spaced laterally adjacent within thebiological sample but within the focal plane of the imaging optics. 7.The light sheet microscope as in claim 2, wherein by moving thecontainer holding the fluid in the z-direction, the sample holder andbiological sample may be held aloft of the container and fluid, suchthat the biological ample can be manipulated without affecting any oneof the focal plane, the detector, the imaging optics, the light sheet ortouching the fluid.
 8. The light sheet microscope as in claim 2, whereinthe sample holder is moved without changing a relative orientation ofthe imaging optics or the focal plane or the light sheet.
 9. The lightsheet microscope as in claim 2, wherein the sample holder is configuredto allow the biological sample to be manipulated without changing arelative orientation of the imaging optics or the focal plane or thelight sheet.
 10. The light sheet microscope as in claim 2, furthercomprising an imaging lens structure upon which the imaging optics ismounted, wherein the imaging lens structure is also movable in thez-direction and wherein the imaging optics are configured to form theimage on the detector, wherein the lens structure, the container, thesample holder, the biological sample, and first and second stages areall movable without changing the focal plane.
 11. The light sheetmicroscope as in claim
 2. wherein the detector comprises: a pixelateddetector wherein the imaging optics form a pixelated image of the focalplane on the pixelated detector, and wherein the imaging optics may bemoved without moving the pixelated detector, the biological sample orthe container.
 12. The light sheet microscope as in claim 2, wherein theimaging optics may be submerged in the fluid by moving the lensstructure downward in the z-direction on the movable second stage or bymoving the first stage and container upward in the z-direction on themovable first stage, and wherein the fluid is a clearing fluid thatrenders the biological sample more transparent to radiation.
 13. Thelight sheet microscope as in claim 2, wherein the sample can be removedand replaced by another sample without moving the focal plane or thelens structure.
 14. The light sheet microscope as in claim 2, wherein atleast a portion of the imaging optics is submerged in the fluid abovethe sample and sample stage to image the irradiated sample.
 15. Thelight sheet microscope as in claim 2, further comprising: a movablethird stage coupled to the sample holder, configured to move thebiological sample in the sample holder in the z-direction to illuminatemultiple depths in the biological sample with the plurality of lightbeams.
 16. The light sheet microscope of claim 15, wherein the lightsheet moves in the z-direction to irradiate multiple areas at differentdepths within the sample by moving the third stage in the z-direction,without affecting the position of the first stage or second stage. 17.The light sheet microscope of claim 16, wherein the different depthscomprise at least 5 different depths.
 18. The light sheet microscope ofclaim 15, wherein the sample holder on the movable third stage, the lensstructure on the movable first stage, and the container on the movablesecond stage can all move independently of, and without disturbing, thefocal plane.
 19. The light sheet microscope of claim 15, wherein themovable third stage has a throw of about 20-40 mm in the z-direction,and 70-90 mm laterally, orthogonal to the z-direction, and the movablefirst stage has a throw of about 40-50 mm in the z-direction.
 20. Thelight sheet microscope as in claim 1, wherein the biological sample isreplaced with a new biological sample, without disturbing the samplestage, the lens structure, or the focal plane, by moving the containeron the movable first stage to expose the biological sample.